All organisms must coordinately regulate their genes during normal development and to prevent disease states. However, the mechanisms by which genes are identified for coordinate regulation remain poorly understood. Our long-term goal is to describe the macromolecular interactions that are critical to target genes for coordinate regulation, the key initial step in their regulation. Dosage compensation is one of the best model systems for studying this process because all of the genes on a single chromosome are specifically identified and co-regulated. Drosophila, like mammals, increase the transcript levels of a large number of diversely regulated genes along the length of the single male X-chromosome precisely two-fold relative to each female X-chromosome [2]. The objective of this application is to generate the first three-dimensional model of how dosage compensation in Drosophila is established, the critical first step in the regulatory process. The Drosophila Male Specific Lethal (MSL) complex is central to dosage compensation; it first identifies the X chromosome using a combination of cis-acting DNA sequences and cotranscriptional recruitment by its roX (RNA on X) non-coding RNA components, and then spreads into the bodies of active genes. However, we do not know how the MSL complex specifically identifies the MSL Recognition Element (MRE) sequences on the male X because known MSL components are insufficient for direct recognition of MREs in vitro. We performed an innovative genetic screen for new regulators of dosage compensation that function in both males and females and thereby identified the previously-unstudied CLAMP zinc-finger protein. Guided by strong preliminary data, we propose the following novel mechanism for identifying genes for coordinate regulation: Concentration of the CLAMP protein to foci within the nucleus initially targets MSL complex to the X- chromosome, followed by synergistic interactions between the two factors that increases their occupancy. The rationale for this work is that our identification f the CLAMP protein provides a critical opportunity to understand the macromolecular interactions required to define a sub-nuclear domain of enhanced transcription. We will test this novel mechanism using two new specific aims: Aim #1: Define how seed sites on the X-chromosome are structured in three dimensions within the nucleus by using Chromosome Conformation Capture; Aim #2: Determine the dynamics of CLAMP localization within the nucleus during early embryogenesis in three dimensions using multiphoton microscopy. Our proposed research is significant because we expect to generate the first three-dimensional model of how an active domain of transcription on an entire chromosome is formed. Our three-dimensional model will allow us to distinguish between two longstanding mechanisms for how a sub-nuclear domain is formed: 1) Discontinuous spreading that involves three-dimensional clustering of initial seed sites; 2) Continuous spreading that involves scanning along the length of the X-chromosome. Defining how CLAMP generates a domain of enhanced transcription is likely to provide key insight into how genes are identified for coordinate regulation across species.